Lecture 6 - Bioc325 Calcium Signaling - 2023 PDF

1. Calcium Signaling 2. Proteins associated with GPCR: a. RGS (Regulators of G-Protein signaling) b. GRK (G-Receptor Kinases)  Understand the vital role of Calcium as a second messenger in signal transduction  Study the regulators of G-Proteins and their role in modu...

1. Calcium Signaling 2. Proteins associated with GPCR: a. RGS (Regulators of G-Protein signaling) b. GRK (G-Receptor Kinases)  Understand the vital role of Calcium as a second messenger in signal transduction  Study the regulators of G-Proteins and their role in modulating GPCR signaling An experiment performed in London nearly 130 years ago, marked the beginning of the calcium signaling saga 1883, Sydney Ringer, was studying contraction of rat hearts. Suspended the hearts in saline made with London tap water (hard), Hearts contracted beautifully. Repeated the experiment with saline made from distilled water, beating of the hearts was weak. Added calcium, hearts maintained contraction. Paved the way to the concept that Ca2+, which was already known to be an important component of bones and teeth, is also a fundamental carrier of messages. Carafoli E, Nature Review MolCell Biol 4:326, 2003  Calcium As a 2nd Messenger At rest: Low Ca2+ levels in the cytoplasm Following specific signals, Ca2+ concentrations in the cytoplasm increase rapidly Ca2+ homeostasis within the cell is maintained by: Ryanodine receptor channels in ER IP3 receptor channel in ER Ca2+ channels on plasma membrane Ca2+ ATPase on plasma membrane Na+/ Ca2+ Exchanger on plasma membrane Gαq Signaling Pathways and Calcium  Ca2+ release Calmodulin Calmodulin dependent effects independent effects Calmodulin: Ca2+-binding protein  First step, binding of Ca2+ to specific proteins to carry the message forward ◦ 2 domains for Ca2+ binding within proteins 1. EF-hand motif 2. C2 domain  Role of EF-hand motif In Calcium Binding Proteins Calmodulin (CaM) and Troponin C (isoform) are the major Ca2+ sensing proteins  CaM: ◦ A ubiquitous 17 kDa-protein ◦ can activate more than 100 enzymes ◦ 4 EF-hand motifs, binds 4 Ca ions ◦ No intrinsic catalytic activity Ca2+ /calmodulin binds to target proteins, including: protein kinases (Ca2+ calmodulin-dependent kinases; CaM-kinases) adenylyl cyclases and phosphodiesterases causing conformational changes and activation of these proteins Calcium binding protein: Calmodulin 2 EF hand motifs Target protein 2 EF hand motifs Molecular Biology of the Cell; Chapter 15; Part 2 A. Ca2+-free Calmodulin (Inactive) ◦ Central helix is shielded by terminal helices ◦ Unable to bind/interact with its targets Central Helix Shifman J M et al. PNAS 2006;103:13968-13973 B. Ca2+-bound Calmodulin (active) ◦ Ca2+ atoms are shown as yellow spheres ◦ Ca2+ binding induces conformational changes ◦ Exposing hydrophobic patches, which are important for target recognition, (shown in red) ◦ Also exposing the central α helical segment Shifman J M et al. PNAS 2006;103:13968-13973 C. Ca2+/Calmodulin bound to target protein (ex: phosphorylase kinase, shown in pink) ◦ Central α helix unwind and form a hinge ◦ Allowing the molecule to bend around the target ◦ N and C terminals approach each other and interact through their hydrophobic regions ◦ Resembles 2 hands holding a rope Shifman J M et al. PNAS 2006;103:13968-13973  Attachment of CaM to its target proteins leads to their conformational changes  three different activation patterns ◦ exposing the catalytic site ◦ changing the target conformation ◦ assembling the structure of the target (inducing its dimerization) A. Relieves auto-inhibition domain (AID) Examples : CaM kinases and calcineurin Cell, Volume 108, Issue 6, 739-742, 22 March 2002  Stages of CaM-kinase Activation Calmodulin-dependent kinase II (CaM Kinase II): Four different activity states based on a combination of protein binding, ion binding, and phosphorylation state Molecular Biology of the Cell Chapter 15 Part 2  Include ◦ Phosphorylase kinase ◦ Myosin light chain kinase ◦ CaM kinases I-IV  Each interacts with CaM  Converts Ca2+ signal into a phosphorylation signal B. Remodels the active site inducing an active conformation example: anthrax adenylyl cyclase Cell, Volume 108, Issue 6, 739-742, 22 March 2002 C. Induces dimerization of K+ channels. Cell, Volume 108, Issue 6, 739-742, 22 March 2002 RGS or Regulators of G-protein Signaling  Nomenclature: Regulator of G-protein Signaling  Equivalent to GTPase-activating-protein (GAP) for small G-proteins  G-protein has an intrinsic GTPase activity = slow rate of GTP hydrolysis  Regulation by RGS = Negatively regulate G-protein signaling 1. Block GDP dissociation 2. Accelerates the GTP hydrolysis  Substrate of RGS = GTP-Ga  RGS activate GTPases for heterotrimeric Gα-subunits 1. RGS increases rate of GTP hydrolysis, shutting down the system 2. RGS can also attenuate G protein actions that are mediated by βγ subunits, by altering the availability of βγ subunit: RGS Mechanism: RGS enhances the affinity of Gα subunits for the βγ after GTP hydrolysis 3. RGS increases the rate of reformation of the hetero- trimer. GPCR stimulate GTP binding, RGS enhance GTP hydrolysis   There are over 50 RGS proteins identified with a variety of RGS activities and sequences.  Crystal structure shows contact residues that are highly conserved between RGS and Ga 100 aa Zheng B et al, Trends Biochem Sci 1999 GRK or G-Receptor Kinases  Ser/Thr Kinases that phosphorylate the agonist – occupied receptor  Leads to the uncoupling of the G-protein from the receptor to modulate its activity  Seven types  Examples: ◦ Rhodopsin kinase or GRK-1 ◦ The beta-adrenergic receptor or βARK-1 and 2 (GRK 2 and 3 respectively) β2-adrenergic receptor Phosphorylation sites for PKA Phosphorylation sites for GRK2 β2-adrenergic receptor kinase  N terminal C terminal GPCR Binding Membrane anchoring RGS binding Catalytic domain CaM binding for Prenylation or (GRK 4, 5, 6) palmitolation for (GRK 1, 4, 6, 7) Gβγ binding for (GRK 2, 3)  Phosphorylated on Ser/Thr residues, by kinases such as PKC  Activated by binding the agonist-occupied receptor  Can be regulated by calcium binding protein (CaM) or lipids  No consensus sequence for GRK but a general pattern  No selective inhibitor L L L Ga Ga GRK -Parr2 [agonist] G coupling GRK arrestin RF RB R* RP Rarr desensitization Will be discussed in the next lecture Proteins Associated with GPCRs-part2: β-arrestins AP-2 Clathrin Dynamin Desensitization, Endocytosis, and Recycling of GPCRs